Cyclodextrin liposomes encapsulating pharmacologic compounds and methods for their use

ABSTRACT

Liposomes containing cyclodextrin in the encapsulated aqueous phase are useful for encapsulation of biologically active substances, especially those which are hydrophilic. The encapsulated cyclodextrin facilitates a slow, controlled release of pharmacologic compounds from the liposomes. The novel methods of the present invention allow the treatment of a variety of pathophysiological states by administering the cyclodextrin-containing liposomes encapsulating the pharmacologic compounds. The present invention also provides a novel method of extending the half life of a pharmacologic compound in an animal.

This application is a 371 of PCT/US94/04,490 filed Apr. 22, 1994 and aContinuation-in-Part application of U.S. Ser. No. 08/051,135, filed Apr.22, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of liposometechnology and pharmacotherapy. More specifically, the present inventionrelates to novel liposomes encapsulating pharmacologic compounds andcyclodextrins and to methods for their use.

2. Description of the Related Art

Liposomes are artificial lipid or phospholipid vesicles enclosingaqueous internal chambers into which molecules, e.g., drugs, can beencapsulated with the intention of achieving slow release of the drugafter administration of the liposome to an individual. In recent years,several types of liposomes have been described (U.S. Pat. No. 4,552,803to Lenk; U.S. Pat. No. 4,310,506 to Baldeschwieler; U.S. Pat. No.4,235,871 to Papahadjopoulos; U.S. Pat. No. 4,224,179 to Schneider; U.S.Pat. No. 4,078,052 to Papahadjopoulos; U.S. Pat. No. 4,394,372 toTaylor; U.S. Pat. No. 4,308,166 to Marchetti; U.S. Pat. No. 4,485,054 toMezei; and U.S. Pat. No. 4,508,703 to Redziniak; Szoka, et al., 1980,Ann. Rev. Biophys. Bioeng. 9:465-508; Liposomes, Marc J. Ostro, Ed.,Marcel-Dekker, Inc., New York, 1983; Poznansky and Juliano, Pharmacol.Rev. 36:277-236, 1984: Kim, et al, Biochim. Biophys. Acta 728:339-348,1983; Kim et al., Biochim. Biophys. Acta 646:1-10, 1981). Unilamellarliposomes have a single bilayer membrane enclosing an aqueous volume(Huang, 1969, Biochemistry 8:334-352) while multilamellar liposomes havenumerous concentric membranes (Bangham et al, 1965, J. Mol. Biol.13:238-252). Multivesicular liposomes are different from eitherunilamellar or multilamellar liposomes in that multivesicular liposomeshave multiple non-concentric aqueous chambers (Kim et al., 1983,Biochim. Biophys. Acta 728:339-348).

Liposome delivery systems have been proposed for a variety ofpharmacologically active compounds including antibiotics, hormones andanti-neoplastic agents (Liposomes, 1983, Marc J. Ostro, Ed.,Marcel-Dekker, Inc., New York, 1983). The use of liposomes toencapsulate pharmacologic agents and the efficacy of liposomal deliverysystems differs according to the water-and lipid-solubility of the drug.For example, hydrophilic substituted for encapsulation in multivesicularliposomes. In contrast, hydrophobic, water insoluble compounds tend tobe incorporated into the lipid bilayer. These compounds, therefore, arenot well suited for encapsulation into the aqueous internal chambers ofa liposome delivery system. The cyclodextrin class of compounds,especially β-cyclodextrin, has been used successfully to solubilizewater-insoluble hydrophobic compounds (Strattan, January 1992, Pharm.Tech. 68-74; Strattan, February 1992, Pharm. Tech. 52-58; Stern, DN&P,2:410-415, 1989; Pagington, Chem. Brit. 23:455-458, 1987).

Encapsulation of water-soluble pharmacologic compounds such asmethotrexate into a variety of drug delivery systems has been previouslyreported. However, the release rates of methotrexate were found to berapid and the previous encapsulations did not result in any majorchanges in pharmacokinetics. Kimelberg et al. reported the half-life ofthe liposomal methotrexate preparation in the cerebrospinal fluid to beextremely short (less than 1 hour) and not significantly different fromthe unencapsulated drug.

Many investigators have attempted to target pharmacologic agents, e.g.,antineoplastic drugs such as methotrexate to a tumor with the intentionof reducing systemic toxicity and increasing tumor kill. One approach isto instill the drug directly into a tumor-containing cavity such asperitoneal cavity or subarachnoid space. However, such intracavitarytherapy is not always successful. One of the problems is that theintracavitary clearance is rapid, resulting in a short drug exposure.For a cell-cycle phase specific drug like methotrexate, prolongedexposure is necessary for optimal efficacy.

The prior art remains deficient in the lack of an effective liposomaldelivery system for some water soluble and biologically active compoundsthat are released too rapidly from liposomes to be practical and useful.The prior art is also deficient in the lack of effective methods for thecontrolled release of such compounds.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there is provided a liposomecomposition, comprising a water soluble compound encapsulated in saidliposome, wherein said liposome composition contains encapsulatedcyclodextrin.

In another embodiment of the present invention, there is provided amethod of treating a pathophysiological state in an individualcomprising administering a liposome composition to the individual, saidcomposition comprising a pharmacologically effective amount of a watersoluble compound encapsulated in said liposome, wherein said liposomecomposition contains encapsulated cyclodextrin.

In yet another embodiment of the present invention, there is provided amethod of increasing the half-life of a compound in an animal comprisingthe step of administering an admixture of liposomes encapsulating thecompound, wherein said liposome encapsulates cyclodextrin.

Other and further objects, features and advantages will be apparent fromthe following descriptions of the presently preferred embodiments in theinvention which are given for the purpose of disclosure and when takenin conjunction with the accompanying drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term "multivesicular liposomes" as used throughout the specificationand claims means man-made, microscopic lipid-vesicles consisting ofintersecting lipid bilayer membranes, enclosing multiple non-concentricaqueous chambers and characterized by a neutral lipid separating theleaflets of a bilayer membrane. In contrast, unilamellar liposomes havea single aqueous chamber, and multilamellar liposomes have multiple"onion-skin" type of concentric membranes, in between which areshell-like concentric aqueous compartments.

The term "solvent spherule" as used throughout the specification andclaims means a microscopic spheroid droplet of organic solvent, withinwhich is multiple smaller droplets of aqueous solution. The solventspherules are suspended and totally immersed in a second aqueoussolution.

The term "MVL-CD-MTX" means a formulation containing methotrexateencapsulated into multivesicular liposomes in the presence ofcyclodextrin.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are not necessarily to scale. Certain features of theinvention may be exaggerated in scale or shown in schematic form in theinterest of clarity and conciseness.

FIG. 1 shows the concentrations of methotrexate in cerebrospinal fluid(CSF) after intracisternal injection of 100 μg (0.22 μmol) ofmultivesicular liposomes encapsulating methotrexate and cyclodextrin(MVL-CD-MTX) (closed circle, free; open square, total) or asunencapsulated methotrexate (closed square). Each data point representsmean and standard deviation from three rats.

FIG. 2 shows the amount of methotrexate remaining within the centralnervous system (CNS) after intracisternal injection of 100 μgmethotrexate as MVL-CD-MTX (open circle, total within CNS; closedcircle, within cranial compartment) or as unencapsulated methotrexate(open square, total; closed square. cranial). Each data point representsmean and standard deviation from three rats.

FIG. 3 shows the amount of the unencapsulated methotrexate (opencircles) and methotrexate MVL-CD-MTX (closed circles) recovered from thesubcutaneous injection site.

FIG. 4 shows the plasma concentrations of methotrexate followingsubcutaneous injection of unencapsulated methotrexate (open circles) andMVL-CD-MTX (closed circles).

FIG. 5 shows the per cent Increased Life Span (ILS) as a function of thelog of the administered dose for unencapsulated methotrexate (opencircles) and for MVL-CD-MTX (closed circles).

FIG. 6 shows the volume-adjusted size distribution of the multivesicularliposome formulation of methotrexate, MVL-CD-MTX. Diameters of particleswere measured in groups of 2-μm intervals from a photomicrograph. Thenumber of particles in each size group was multiplied by the cube of theradius to obtain relative volumes of each size category and then dividedby the sum of the relative volumes of all particles to obtain percent oftotal volume represented by each size category. The capture volume was12.9±1.0 μl/μmole of lipids.

FIG. 7 shows the release of methotrexate from MVL-CD-MTX suspended in0.9% NaCl solution kept at 4° C. (shaded circle); in 0.9% NaCl solutionat 37° C. (open circle); and in human plasma at 37° C. (shaded box).Each point is the mean and the standard deviation from threeexperiments. The ordinate scale is logarithmic.

FIG. 8 shows the intraperitoneal concentrations of methotrexate afterintraperitoneal injection of 10 mg/kg (22 μmoles/kg) of methotrexate asunencapsulated methotrexate (open circles), unencapsulated cyclodextrin-methotrexate complex (shaded triangles) or multivesicular liposomeencapsulated methotrexate, MVL-CD-MTX (shaded circles, free; open boxes,total). Each point represents the mean and the standard deviation from agroup of three mice.

FIG. 9 shows the amounts of methotrexate remaining within the peritonealcavity after injection of 10 mg/kg (22 μmoles/kg) of methotrexate asunencapsulated methotrexate (open circles), unencapsulated cyclodextrin-methotrexate complex (closed triangles) or multivesicular liposomeencapsulated methotrexate, MVL-CD-MTX (shaded boxes). Each pointrepresents the mean and the standard deviation from a group of threemice.

FIG. 10 shows the survival curves of mice treated intraperitoneally onday 1 with 0.9% NaCl solution (shaded triangles), 2000 mg/kg ofunencapsulated methotrexate (shaded circles), 2500 mg/kg ofunencapsulated methotrexate (open circles) 15 mg/kg of MVL-CD-MTX(shaded boxes) and 20 mg/kg of MVL-CD-MTX (open boxes).

FIG. 11 shows the increased life time (ILS) versus dose (mg/kg) of micetreated on day 1 with unencapsulated methotrexate (open circles) andwith MVL-CD-MTX (shaded circles). Each data point represents median ILSfrom a group of five mice. Comparison to optimal unencapsulatedmethotrexate dose (2000 mg/kg) was by the Mann-Whitney non-parametrictest: *, p<0.02; **, p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to forming inclusion complexes ofwater-soluble compounds, such as methotrexate, with cyclodextrins,preferably β-cyclodextrin, and to encapsulating the inclusion complexinto liposomes for controlled release. For use in the practice of thisinvention the cyclodextrin preferably forms an inclusion complex withthe water soluble compound wherein the apolar cavity of the cyclodextrinis occupied by or sequesters the compound sufficiently to slow the rateof release from the liposome composition. The rim or the periphery ofthe inclusion complex is hydrophilic with the result that the inclusioncomplex forms a solution in aqueous media. The cyclodextrin-complexedwater soluble substance can then be encapsulated into liposomes.

In addition to preventing incorporation of water soluble compounds intothe lipid layers of the liposomes during their formation, Applicantshave discovered that formation of an inclusion complex results in areduction in the rate of release of the hydrophilic compound from theliposome compared to the rate of release of the same compoundencapsulated in the absence of the cyclodextrin.

The present invention provides a liposome composition, comprising apharmacologically active amount of a biologically active compoundencapsulated in said liposome, wherein said liposome composition furthercontains encapsulated cyclodextrin. Preferably, the biologically activecompound is water soluble. In the practice of this invention, the watersoluble compound generally has water solubility of greater than about 1μg/ml, preferably greater than about 100 μg/ml, and most preferablygreater than about 1 mg/ml, in the absence of cyclodextrin.

As used herein, the term "pharmacologic" or "pharmacologically active"is used interchangeably with "biological" or "biologically active".

Cyclodextrins are chiral, toroidal-shaped molecules formed by the actionof the enzyme cyclodextrin transglycosylase on starch. These cyclicoligomers contain from 6 to 12 glucose units bonded throughα-(1,4)-linkages. The three smallest homologs, α-cyclodextrin,β-cyclodextrin and γ-cyclodextrin are available commercially; largerhomologs must be produced and isolated individually. The secondary 2-and 3-hydroxy groups line the mouth of the cyclodextrin cavity and havea staggered orientation. The primary 6-hydroxyls are at the opposite endof the molecule. The inside of the cyclodextrin cavity is relativelyhydrophobic since all hydroxyls are directed toward the outside of themolecule.

It is specifically contemplated that many different types ofcyclodextrins would be useful in the compositions and methods of thepresent invention. For example, the present invention may use naturalα-, β- or γ cyclodextrins. Similarly, the present invention may utilizesemisynthetic substituted cyclodextrins such as; methyl cyclodextrins,ethyl cyclodextrins, hydroxyethyl cyclodextrins, hydroxypropylcyclodextrins, branched cyclodextrins, cyclodextrin polymers ormonosuccinyl dimethyl β-cyclodextrin. Most preferred for thecompositions and methods of the present invention is2-hydroxypropyl-β-cyclodextrin.

Generally, the concentration of cyclodextrin used in preparing theliposomes of the present invention is that which slows the release of apharmacologic compound from the liposome after administration to ananimal. Preferably, the cyclodextrin is present in the liposomecomposition in an amount of from about 10 milligrams per ml to about 400milligrams per ml. More preferably, the amount of cyclodextrin in theliposome is about 100 mg/ml.

Generally, the liposome of the present invention may be any that whenprepared with encapsulated cyclodextrin provides slow, controlledrelease of pharmacologic compounds. Preferably, the liposome is selectedfrom the group of unilamellar, multilamellar and multivesicularliposomes. Most preferably, the liposome is a multivesicular liposome.

Generally, the biologically active compound encapsulated in the liposomeoil the present invention may be any whose release rate from a liposomeencapsulating cyclodextrin is slower than that in the absence of thecyclodextrin. Therapeutic biologically active compounds may be selectedfrom the general group consisting of anti-neoplastic agents,anti-infective agents, anti-depressives, antiviral agents,anti-nociceptive agents, anxiolytics and hormones.

Representative examples of anti-neoplastic agents useful in thecompositions and methods of the present invention include methotrexate,taxol, tumor necrosis factor, chlorambucil, interleukins, bleomycin,etoposide, fluorouracil and vinblastine.

Representative examples of anti-infective agents useful in thecompositions and methods of the present invention include pentamidine,metronidazole, penicillin, cephalexin, tetracycline and chloramphenicol.

Representative examples of anti-viral agents useful in the compositionsand methods of the present invention include dideoxyoytidine,zidovudine, acyclovir, interferons, dideoxyinosine and ganciclovir.

Representative examples of anxiolytics and sedatives useful in thecompositions and methods of the present invention includebenzodiazepines such as diazepam, barbiturates such as phenobarbital andother compounds such As buspirone and haloperidol.

Representative examples of hormones useful in the compositions andmethods of the present invention include estradiol, prednisone, insulin,growth hormone, erythropoietin, and prostaglandins.

Representative examples of anti-depressives useful in the compositionsand methods of the present invention include fluoxetine, trazodone,imipramine, and doxepin.

Representative examples of anti-nociceptives useful in the compositionsand methods of the present invention include hydromorphine, oxycodone,fentanyl, morphine and meperidine.

The list of therapeutic biologically active agents described above isonly exemplary and not meant to limit the scope of the present inventionin any fashion. Many other classes of pharmacologic agents would beuseful in the compositions and methods of the present invention,including local anesthetics, vitamins, vaccines, wound healingstimulators, immunosuppressives, anti-emetics, anti-malarial agents,anti-fungal agents, anti-psychotics, anti-pyretics, coagulants,diuretics, calcium channel blockers, bronchodilatory agents, etc.

The present invention also provides a method of increasing the half-lifeof a pharmacologic compound in an animal comprising the step ofadministering an admixture of liposomes encapsulating the pharmacologiccompound, wherein said liposome further encapsulates cyclodextrin.

The present invention additionally provides a method of treating apathophysiological state in an individual comprising administering aliposome composition to the individual, said composition comprising atherapeutically effective amount of a compound encapsulated in saidliposome, wherein said liposome composition further encapsulatescyclodextrin. The term "therapeutically effective" as it pertains to thecompositions of the invention means that biologically active therapeuticagent is present in the aqueous phase within the vesicles at aconcentration sufficient to achieve a particular medical effect forwhich the therapeutic agent is intended. Examples, without limitation,of desirable medical effects that can be attained are chemotherapy,antibiotic therapy, and regulation of metabolism. Exact dosages willvary depending upon such factors as the particular therapeutic agent anddesirable medical effect, as well as patient factors such as age, sex,general condition, and the like. Those of skill in the art can readilytake these factors into account and use them to establish effectivetherapeutic concentrations without resort to undue experimentation.

Generally, however, the dosage range appropriate for human use includesthe range of 0.1-6000 mg/sq m of body surface area. For someapplications, such as subcutaneous administration, the dose required maybe quite small, but for other applications, such as intraperitonealadministration, the dose desired to be used may be very large. Whiledoses outside the foregoing dose range may be given, this rangeencompasses the breadth of use for practically all the biologicallyactive substances.

The liposomes of the present invention may be administered by anydesired route. For example, administration may be intrathecal,intraperitoneal, subcutaneous, intramuscular, intravenous,intralymphatic, oral and submucosal. Administration may also be todifferent kinds of epithelia including the bronchiolar epithelia, thegastrointestinal epithelia, the urogenital epithelia and various mucousmembranes in the body. As one skilled in the art will appreciate, thebest route of administration may depend upon the biologically activecompound selected. For instance, although methotrexate can be givenorally, parenteral administration has certain advantages. The absorptionrate of methotrexate after oral administration is highly variable amongpatients and appears to be saturable. In contrast, absorption of thedrug after im or sc administration is much more predictable andcomplete, resulting in higher serum concentrations than after an oraldose.

Cyclodextrin-containing liposomes are useful in extended-release drugdelivery of subcutaneously administered pharmacological agents forseveral reasons. They are quite stable in storage. Moreover, the drugcan be released over extended time periods, both in vitro and in vivo.Their sponge-like internal structure, results in efficient encapsulationinto a chambers, stability in storage, and extended release in vivo. Forinstance, the half-life in plasma of methotrexate can be increased by206-fold over that of free methotrexate, and with peak plasmaconcentration was 126-fold lower compared to unencapsulatedmethotrexate. As a consequence of the significant modifications of thepharmacokinetics achieved by encapsulation of a drug encapsulated in theliposome in the presence of cyclodextrin, drug potency can be increasedby over 100 fold. For instance the potency of methotrexate can beincreased by 130 fold through administration in accordance with theteachings of this invention, and LD₅₀ can be decreased 110 fold. Thesechanges in potency and LD₅₀ indicate no significant change intherapeutic index due to introduction into the liposomes duringencapsulation of the biologically active compound.

The liposomal compositions of the present invention may be modified toimpart organ or cellular targeting specificity. These modifications maybe particularly relevant when using the liposomal compositions of thepresent invention to administer pharmacologic agents that are highlytoxic or that produce severe side effects.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, e.g., organ-specific, cell-specific or organelle-specific.Mechanistic targeting can be distinguished based upon whether it ispassive or active. Passive targeting utilizes the natural tendency ofliposomes to distribute to cells of the reticulo-endothelial system inorgans which contain sinusoidal capillaries. Active targeting, incontrast, involves alteration of the liposome by coupling the liposometo a specific ligand such as a monoclonal antibody, sugar, glycolipid,protein or by changing the composition or size of the liposome in orderto achieve targeting to organs and cell types other than the naturallyoccurring sites of localization. See, e.g., Remington's PharmaceuticalSciences, Gannaro, A. R., ed., Mack Publishing, 18th edition, pp.1691-1693. For instance, MVL-CD-MTX particles can be synthesized inlarge average sizes to decrease their uptake into lymphatics andsystemic circulation after injection into body cavities or into tissuespaces, such as subcutaneous space. Their large size may also inhibituptake into macrophages.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal composition of the presentinvention, lipid groups may be incorporated into the lipid bilayer ofthe liposome in order to maintain the targeting ligand in stableassociation with the liposomal bilayer. Various linking groups can beused for joining the lipid chains to the targeting ligand. The compoundsbound to the surface of the targeted delivery system may vary from smallhaptens of from about 125-200 molecular weight to much larger antigenswith molecular weights of at least 6000, but generally of less than 1million molecular weight. The following examples are given for thepurpose of illustrating various embodiments of the methods of thepresent invention and are not meant to limit the present invention inany fashion.

EXAMPLE 1 Synthesis of MultivesicularLiposome-Methotrexate-βCyclodextrin Formulation, MVL-CD-MTX

Multivesicular liposomes encapusulating methotrexate in the presence ofcyclodextrin (MVL-CD-MTX) were prepared using a method described by Kimet al (Cancer Treat. Rep. 71:705, 1987) with some modifications.Briefly, for each batch of MVL-CD-MTX, the discontinuous aqueous phaseconsisted of 2-hydroxypropyl-β-cyclodextrin solution (100 mg/ml), HCl(0.1N) and methotrexate (10 mg/ml). One ml of the discontinuous aqueousphase was added into a one dram vial containing 13.9 μmol dioleoyllecithin, 3.15 μmol dipalmitoyl phosphalidy/glycerol, 22.5 μmolcholesterol, 2.7 μmol triolein and 1 ml chloroform. The vial wasattached horizontally to the head of a vortex mixer and shaken atmaximum speed for 6 minutes. One-half of the resulting "water-in-oil"emulsion was expelled rapidly through a narrow-tip Pasteur pipette intoeach of two 1-dram vials, each containing 2.5 ml water, glucose (32mg/ml) and free-base lysine (40 μM). Each vial was then shaken on thevortex mixer for 5 seconds at maximum speed to form chloroformspherules. The chloroform spherrule suspensions in the two vials weretransferred into a 250-ml Erlenmeyer flask containing 5 ml water,glucose (32 mg/ml), and free base lysine (40 mM). A stream of nitrogengas at 7 liter per minute was used to evaporate the chloroform over a10-15 minute period a 37° C. The MVL-CD-MTX particles were then isolatedby centrifugation at 600×g for 5 minutes and washed three times with0.9% NaCl solution.

EXAMPLE 2 Intrathecal pharmacokinetic studies

Rats were anesthetized with ketamine HCl (90 mg/kg and acetopromazinemaleate (2.2 mg/kg, injected intramuscularly) and mounted in aconventional stereotaxic frame. Using a No. 15 blade, a midlinecutaneous incision approximately 1 cm in length was made from theoccipital crest to just behind the ears. The muscle ligament along theoccipital crest at the skull was detached with a scalpel for 4 mm oneither side of the midline. Using both the sharp and blunt ends of aperiosteal elevator, the muscle from the occipital bone was freed downto the atlanto-occipital membrane. A retractor was placed in theincision to draw the muscle aside and obtain a clear view of theatlanto-occipital membrane. Either 20 μl unencapsulated methotrexate or20 μl MVL-CD-MTX in 0.9% NaCl, both containing 100 μg (0.22 μmol)methotrexate, was then injected over 20 seconds via a 30-gauge needlethrough the membrane. The needle was withdrawn, the skin was suturedwith 3-0 silk, and the animal was given 10 ml lactated Ringer'solutionsubcutaneously for hydration.

At appropriate time points after injection, the atlanto-occipitalmembrane was again exposed under anesthesia and a sample ofcerebrospinal fluid ranging from 30 to 60 μl was obtained through a19-gauge needle. Cerebrospinal fluid samples were obtained from threerats at each time: at 1 minutes and at 4, 24, and 48 hours afterinjection in the unencapsulated methotrexate group and at 1 minutes andat 1, 3, 7, 14 and 21 days after injection in the Depo-methotrexategroup. The CSF samples from the MVL-CD-MTX group were diluted with 70 μl0.9% NaCl solution and then immediately centrifuged in an EppendorfMicrofuge for 1 minute to separate a supernatant containing releasedfree methotrexate from a pellet containing encapsulated methotrexate.Next, 50 μl of methanol and 50 μl of sterile water were sequentiallyadded to the pellet and vortexed to break the MVL-CD-MTX particles. Thecerebrospinal fluid samples were then kept frozen at -20° C. untilanalyzed using a high-performance liquid chromatography (HPLC) system asdescribed below.

After cerebrospinal fluid sampling, the animals were sacrificed with anoverdose of ketamine (90 mg/kg) and acetopromazine (20 mg/kg), injectedintraperitoneally. Blood samples were obtained via cardiac puncture andthorough exsanguination was performed. The plasma was separated and keptfrozen at -20° C. until analyzed by Emit methotrexate assay on COBASFara instrument (Roche Diagnostic Systems). The calvarium was thenexposed and carefully removed with a bone rongeur. The entire content ofthe cranial compartment was collected by scooping out the exposed brainwith a spatula and washing the cranial vault thoroughly with distilledwater. The spinal compartment content was then collected separately; thespinal cord was extruded forward into the cranial vault by pushingdistilled water rapidly through a 19-gauge needle inserted into thelower lumbar spinal canal at a point 2.5 cm rostral to the origin of thetail. The empty spinal canal was washed out thoroughly with distilledwater to complete collection of methotrexate in the spinal canal. Thecranial compartment samples were analyzed separately from the spinalcanal samples. Both tissue samples were homogenized with water using aPolytron homogenizer.

EXAMPLE 3 Measurement of methotrexate

The amount of methotrexate in the spinal compartment was calculated byadding the amount from the cisternal cerebrospinal fluid sample. Thehomogenized samples of brain or spinal cord were analyzed with HPLCafter extraction as described by Alkayal et al. Ther. Drug Monit. 12:191(1990). Briefly, in a glass centrifuge tube, a 500 μl aliquot ofhomogenate, 100 μl of theophylline aqueous solution (internal standard,2.0 mg/ml), 250 μl trichloroacetic acid solution (10% in water), and 250μl glacial acetic acid were placed and mixed. Then, methotrexate freeacid was extracted with 5 ml ethyl acetate. Ethyl acetate organic phasewas decanted and evaporated under nitrogen at 60° C. The extractedresidue was dissolved in 200 μl of mobile phase and 100 μl of theresulting solution was injected into the HPLC. Mobile phase consistingof H₃ PO₄ (10 mM): methanol in 180:540:280 ratio (final pH of 3) waspumped at a flow rate of 1 ml/min with a Waters model 510 pump through aBeckman ultrasphere ODS 5 μm×4.6 mm×25 cm column (Beckman, Carlsbad,Calif.). Methotrexate was detected at 303 nm with a Waters Model 490programmable multiwavelength detector (Waters Assoc., Milford, Mass.).The retention times of theophylline and methotrexate were 5.2 minutesand 7.2 minutes, respectively. The limit of detection was 5 pmol ofmethotrexate injected.

EXAMPLE 4 Pharmacokinetic analysis

The RSTRIP computer program (MicroMath Scientific Software, Salt LakeCity, Utah) was used to perform the pharmacokinetics analysis. The areaunder the curve (AUC) was determined by linear trapezoidal rule up tothe last measured concentration and extrapolated to infinity.

EXAMPLE 5 Characterization of MVL-CD-MTX

The average volume-weighted diameter of MVL-CD-MTX was found to be14.1±3.4 (±standard deviation, SD). Encapsulation efficiency was 64.5±6%(n=6) and captured volume was 12.9±1.0 μl/umol of lipids. Storage ofMVL-CD-MTX at 4° C. in 0.9% NaCl solution resulted in less than 5%release of methotrexate after 4 months.

EXAMPLE 6 CNS Pharmacokinetics

FIGS. 1 and 2 compare the central nervous system (CNS) pharmacokinetics(in terms of CSF concentration and CNS amount) for MVL-CD-MTX andunencapsulated methotrexate. The CSF concentration of free methotrexatereached a maximum on day 1 and then decreased in a biexponential fashionwith initial and terminal half-lives of 0.41 and 5.4 days, respectively.The terminal half-life was 18 times longer than that for unencapsulatedmethotrexate.

Following injection of MVL-CD-MTX, the total amounts of drug within CNSdecreased with a half-life of 9 days compared to 0.03 days forunencapsulated methotrexate. At the end of the 21-day period, 18% of themethotrexate remained within the CNS after MVL-CD-MTX injection.

Pharmacokinetic parameters for methotrexate and MVL-CD-MTX within theCNS are summarized in Table 1. Maximum concentration of freemethotrexate after MVL-CD-MTX administration was about 70 times lowerthan that after administration of unencapsulated methotrexate. Theproportion of the total amount of methotrexate within the cranialcompartment were 12±8%, 65±11%, 51±40%, and 65±36%, respectively at 1minute and 7, 14 and 21 days after injection of MVL-CD-MTX and 4±1% and23±1%. respectively, at 1 minute and 4 hours after injection ofunencapsulated methotrexate.

                  TABLE 1                                                         ______________________________________                                        Pharmacokinetics parameters of methotrexate                                   in the CNS after a 100 μg injection                                                 Unencapsulated                                                                          MVL-CD-MTX                                                          Methotrexate                                                                            Free      Total                                            ______________________________________                                        C.sub.max (μM)                                                                        1751 ± 302                                                                             23.7 ± 11.7                                                                          1133 ± 631                                Conc. t.sub.1/2 α (days)                                                           0.024       0.41      0.18                                         Conc. t.sub.1/2 β (days)                                                            0.30        5.4       4.0                                          AUC (μM × days)                                                                 154.3       50.5      624.2                                        Amount t.sub.1/2 (days)                                                                  0.03        NA        9.0                                          ______________________________________                                         C.sub.max, maximum CSF concentration;                                         t.sub.1/2 ; halflife;                                                         AUC, area under the curve;                                                    NA, not applicable                                                       

Analysis of plasma concentrations showed undetectable levels ofmethotrexate (limits of detection being 0.02 μM) except at one timepoint after unencapsulated drug (4 hours after intracisternal injection:0.11±0.02 μM).

EXAMPLE 7 Toxicities

No abnormalities were observed in the behavior of rats given injectionsof MVL-CD-MTX. Three rats injected with MVL-CD-MTX gained weight from343±5 to 383±19 grams over the 3 weeks. In contrast, control ratswithout any injections or surgical interventions grew from 340±1 to400±12 grams.

The encapsulation of methotrexate in multivesicular liposomes resultedin a 18-fold increase in the terminal half-life of free methotrexatefrom the cerebrospinal fluid. The free methotrexate concentrationsstayed above 0.5 μM, considered the minimal cytotoxic concentrationestimated from studies in vitro, for 7-14 days after a single injectionof MVL-CD-MTX. In contrast, the duration was about 1 day for theunencapsulated drug.

The area under the curve of free concentrations for the MVL-CD-MTX groupwas one third of that for the unencapsulated group. This may beattributable to saturation of the methotrexate cerebrospinal fluidclearance mechanism when high free methotrexate concentrations occur inthe unencapsulated group. A second possibility is that a higher fractionof the free methotrexate penetrates into the brain and spinal cordparenchyma by extended exposure and thus a smaller fraction remains inthe cerebrospinal fluid. Yet another possibility is that the area underthe curve for the MVL-CD-MTX was underestimated due to the samplingschedule.

The comparison of the total amount of methotrexate and the amount withinthe cranial compartment (FIG. 2) showed good distribution of MVL-CD-MTXinto both spinal and cranial compartments after intracisternalinjection. For example, at day 21 the amount of methotrexate within thecranial compartment was only 65±36% of the total (cranial plus spinal)amount. However, a large fraction of methotrexate in the cisternalsample was in the form of free drug after the first day of injectionwith MVL-CD-MTX. A high density of MVL-CD-MTX particles relative to thecerebrospinal fluid may result in settling of MVL-CD-MTX particles bygravity away from the cisternal cerebrospinal fluid, whereas thereleased free methotrexate is free to diffuse. The extended release ofmethotrexate from MVL-CD-MTX, both in vitro and in vivo, indicates thatmultivesicular liposomes would be useful as a drug depot formethotrexate.

With MVL-CD-MTX, neurotoxicity can be reduced by keeping most of theinitial bolus of methotrexate within the multivesicular liposomes andyet tumor kill enhanced by maintaining the free methotrexate to justabove the minimum cytotoxic concentration for an extended period. Thepresent invention demonstrates the utility of cyclodextrin liposomes asa slow-releasing drug delivery system for biologically activesubstances, such as methotrexate. The present invention demonstrates theutility of less frequent intra-CSF administration for the prophylaxisand treatment of leptomeningeal leukemia or carcinomatosis in humans.

EXAMPLE 8 Subcutaneous Administration of MVL-CD-MTX

BDF1 and DBA/2J mice were from Simonsen Laboratories, Gilroy, Calif. TheL1210 leukemia was maintained by serial intraperitoneal passage inDBA/2J female mice. MVL-CD-MTX was prepared as described in Example 1.

Subcutaneous (sc) pharmacokinetic studies were done using male BDF1mice, weighing 20-25 grams. Mice were injected sc into the center ofabdominal skin with 10 mg/kg (22 μmoles/Kg) of unencapsulated standardmethotrexate or MVL-CD-MTX in 200 μl of 0.9% NaCl solution, using a30-gauge hypodermic needle. Blood samples were obtained from the jugularvein under anesthesia at time points, 0, 0.25, 1 and 4 hours for theencapsulated methotrexate group and at time points 0, 1, 3, 7, 14 and 21days for the MVL-CD-MTX group. At each time point, 3 animals weresacrificed. The plasma was separated and kept frozen at -20° C. untilanalyzed by Emit^(R) methotrexate assay on COBAS Fara instrument (RocheDiagnostic Systems, Montclair, N.J.).

A full thickness of the abdominal wall tissue, including the entire skinand the underlying peritoneal membrane, was then excised from the costalmargin to the inguinal area and from one flank to the other. The entiretissue specimen was homogenized after addition of at least 20 ml ofdistilled water with a Polytron homogenizer. The homogenate wassonicated for 60 seconds at a maximum setting with a Biosonic IV probesonicator and filtered through a YMT ultrafiltration membrane (AmiconCorp, product #4104). All the samples were kept at -20° C. until assayedby HPLC. The RSTRIP program was used to perform the curve fitting. AUCwas determined by linear trapezoidal rule up to the last measuredconcentration and extrapolated to infinity.

EXAMPLE 9 HPLC assay

A mobile phase consisting of H₃ PO₄ (10 mM): KH₂ PO₄ (10 mM): methanolat 162:488:350 ratio (pH =3) was pumped at a flow rate of 1 ml/min witha Waters Model 510 pump through a Beckman ultrasphere ODS 5 μl 4.6 mm×25cm column. Methotrexate was detected at 303 nm by a UV Waters 490programmable Multiwave-length Detector. Retention time of methotrexatewas 5 minutes and the detection limit was 5 pmols injected.

EXAMPLE 10 Toxity and efficacy studies

BDF1 mice were injected with 10⁶ L1210 cells into the peritoneal cavityon Day 0 and treated sc with a single dose of encapsulated methotrecateoar MVL-CD-MTX suspended in 0.9 % NaCl on Day 1. Five animals were ineach group except for the control (given 0.9% NaCl alone), where 10animals were used. Each animal was observed for survival. Mediansurvival time was used to calculate the "increased life span" (ILS)according to the formula:

    ILS=(T/C)/C×100%

where T is the median survival time of treated groups and C is themedian survival time for control groups.

EXAMPLE 11 MVL-CD-MTX Pharmacokinetics

Pharmacokinetic parameters are summarized in Table 2. After asubcutaneous injection, total amount of methotrexate in skin decreasedexponentially with a half-life of 0.16 hours for unencapsulatedmethotrexate and 50.4 hours for MVL-CD-MTX (FIG. 3). In plasma, thehalf-lives were 0.53 hours for encapsulated methotrexate and 109 hoursfor MVL-CD-MTX. Peak plasma levels were 17.4±5.2 μM (SD) at 15 minutesfor the encapsulated methotrecate and 0.138±0.061 μM (SD) at day 3 forMVL-CD-MTX (FIG. 4).

                  TABLE 2                                                         ______________________________________                                        Pharmacokinetics Parameters                                                   ______________________________________                                        SUBCUTANEOUS                                                                  Amount t.sub.1/2 * (h)                                                                       0.16     50.4                                                  PLASMA                                                                        C.sub.max ± SD (μM)                                                                    17.4 ± 5.2                                                                          0.138 ± 0.061                                      Conc. t.sub.1/2 (h)                                                                          0.53     109                                                   AUC (μM × h)                                                                        17.3     24.5                                                  ______________________________________                                    

EXAMPLE 12 Efficacy Of SC MVL-CD-MTX

FIG. 5 shows the ILS curves in a murine L1210 model. The maximumefficacy (ILS max) was 183% for unencapsulated methotrexate and 217% forMVL-CD-MTX (=0.5 by Mann-Whitney U-Test). The relative potency of thesingle-dose MVL-CD-MTX versus unencapsulated methotrexate was 130 (byPHARM/PCS program, MicroComputer Specialists, Philadelphia, Pa.). TheLD₅₀ was calculated after probit transformation. The LD₅₀ for a singledose of unencapsulated methotrexate was 2650 mg/kg and that forMVL-CD-MTX was 24 mg/kg, a ratio of 110.

The half-life in plasma was 206-fold longer and peak plasmaconcentration was 126-fold lower compared to unencapsulatedmethotrexate, whereas the area under the curve was essentiallyunchanged. As a consequence of the significant modifications of tiepharmacokinetics, drug potency was increased 130 fold and the value ofthe LD₅₀ indicated no significant change in therapeutic index.

EXAMPLE 13 Pharmacokinetics of Peritoneal Cavity Administration In VitroDrug Release Studies

Methotrexate release studies were done by adding a minimum of 40×volumesof 0.9% NaCl solution or human plasma from blood bank to washedMVL-CD-MTX pellets and kept at 4° C. or 37° C. For 37° C. incubation,0.01% sodium azide was added to inhibit growth of microorganisms. Atappropriate time points, aliquots were removed after thorough mixing,diluted with 5-fold volume of 0.9% NaCl solution and centrifuged in anEppendorf microfuge for 1 minute. After the supernatant was removed, 200μl of methanol was added to the pellet to break MVL-CD-MTX particles,and the resulting mixture was stored at -20° C. until analysis. Theamount of methotrexate in the pellet was analyzed by HPLC and wasexpressed as percent of the initial amount remaining as MVL-CD-MTX.Methotrexate was measured by HPLC as described in Example 9.

EXAMPLE 14 Pharmacokinetic studies

The in vivo studies were done on male BDF1 mice weighing 18-25 g. Thegroup of mice was injected ip with 10 mg/kg of methotrexate in 1 ml of0.9% NaCl as unencapsulated methotrexate control,cyclodextrin-methotrexate control (methotrexate 20 mg/m1;2-hydroxypropyl1 -β-cyclodextrin, 2 mg/ml; glucose, 6.4 mg/ml; free-baselysine, 8 mM; and HCl, 2 mM) or MVL-CD-MTX. Three mice were sacrificedand blood samples were collected from the jugular vein and placed in aheparinized tube at 0 hour (immediately after the injection), 1 hour and4 hours after injection of the unencapsulated methotrexate orcyclodextrin-methotrexate complex; and 1, 5, 10 and 20 days afterinjection of MVL-CD-MTX. The plasma was separated and was kept frozen at-20° C. until analyzed by the Emit^(R) methotrexate assay on COBAS FaraInstrument. The Emit^(R) assay is a homogeneous enzyme immunoassaytechnique based on the competition between drug present in the sampleand drug labeled with the enzyme glucose-6-phosphate dehydrogenase forantibody binding sites. The limit of sensitivity was 0.02 μM.

Five μl of the peritoneal fluid samples were collected into a glasscapillary pipette and diluted into 140 μl of 0.9% NaCl solution. For theanimals injected with MVL-CD-MTX, the samples were spun in Eppendorfmicrofuge for 1 minute to separate the supernatant (free methotrexate)and pellet (encapsulated methotrexate). Fifty μl of methanol was addedto the pellet and vortexed to break multivesicular particles. Theperitoneal cavities were then washed out thoroughly with 2-3 ml of 0.9%NaCl solution thrice. All samples were kept frozen at -20° C. untilassayed by HPLC. Extraction of the samples was not necessary, nointernal standard was used and there were no interfering peaks. RSTRIPcomputer program was used to analyze pharmacokinetic data. The areaunder the curve was determined by linear trapezoidal rule up to the lastmeasured concentration and extrapolated to infinity.

EXAMPLE 15 Efficacy and toxicity studies

BDF1 mice were inoculated ip with 10⁶ L1210 cells on Day 0, and treatedon Day 1 with a single ip injection of unencapsulated methotrexate,MVL-CD-MTX, or blank multivesicular liposomes in 1 ml of 0.9NaClsolution. There were five mice per group and fifteen mice were used asuntreated controls (given 1 ml of 0.9% NaCl solution). The result wasexpressed as "increased life span".

EXAMPLE 16 In vitro release studies

The resulting multivesicular liposome particles had a volume-weightedaverage diameter (±SD) of 11.3±3.3 μM (FIG. 6 and 7) and the percentageof capture of 64.5±6.0 % (n=6). Storage of MVL-CD-MTX in 0.9% NaClsolution at 4° C. resulted in less than 5% leakage at 4 months. At 37°C. in 0.9% NaCl solution, there was 63±12% (means±SD) of the initialamount of methotrexate inside the multivesicular liposome particlesafter 3.5 months. In human plasma at 37° C., the half-life of drugrelease was 40 days (FIG. 8).

EXAMPLE 17 Pharmacokinetics

The intraperitoneal pharmacokinetics parameters are summarized in theTable 3. Following ip injection of MVL-CD-MTX, total concentration ofmethotrexate in the peritoneal cavity increased five-fold over the firstday (FIG. 8). During this period of time, the amount of fluid in thecavity decreased significantly. After Day 1, the total concentrationdecreased with a half-life of 1.9 days (FIG. 9).

                  TABLE 3                                                         ______________________________________                                        Pharmacokinetic parameters of methotrexate                                    after intraperitoneal administration                                                 Un-                                                                           encapsulated      MVL-CD-MTX                                                  MTX     CD-MTX    Free      Total                                      ______________________________________                                        PERITONEAL                                                                    Conc. t.sub.1/2.sup.b (h)                                                              0.54      0.46      39.6    45.6                                     Amount t.sub.1/2 (h)                                                                   0.45      0.41      .sup. NA.sup.d                                                                        62.4                                     C.sub.max.sup.c + SD                                                                   430 ± 13                                                                             379 ± 10                                                                             66.7 ± 18.3                                                                        1863 ± 168                            (μM)                                                                       AUC (μM · h)                                                               233       316       12260   273800                                   PLASMA                                                                        Conc. t.sub.1/2 (h)                                                                    0.9       0.6       240     NA                                       Cmax.sup.c + SD                                                                         3.3 ± 0.03                                                                           3.3 ± 0.03                                                                          0.05 ± 0.05                                                                        NA                                       (μM)                                                                       AUC (μM · h)                                                               11.2      12.2      18.4    NA                                       ______________________________________                                         .sup.a cyclodextrin-methotrexate                                              .sup.b halflife                                                               .sup.c peak concentrations                                                    .sup.d not applicable                                                    

EXAMPLE 18 Efficacy studies

FIG. 10 shows the survival curves and FIG. 11 shows the ILS (increasedlife span) curves in the murine L1210 model. The equipotent doses (EPD)appeared to be 6 mg/kg for MVL-CD-MTX and 2000 mg/kg for unencapsulatedmethotrexate calculated at the optimal unencapsulated methotrexate dose.Therefore, MVL-CD-MTX increased potency of single-dose methotrexate 334fold. The maximum efficacy (ILS max) was increased from 100% ILS forunencapsulated methotrexate to 217% ILS for MVL-CD-MTX, more than 2 foldincrease p<0.01 by the Mann-Whitney nonparametric test).

LD₅₀ was calculated after probit transformation by PHARM/PCS program(MicroComputer Specialists, Philadelphia, Pa.). LD₅₀ for a single doseof unencapsulated methotrexate was 2755 mg/kg and that for MVL-CD-MTXwas 17.5. The therapeutic index (TI) for single IP dosage was calculatedby the equation:

    TI=LD.sub.50 /EPD

The TI for unencapsulated methotrexate was 1.4 and that for MVL-CD-MTXwas 2.9. The blank multivesicular liposomes containing glucose and nomethotrexate had no toxic effect on a group of five mice without tumors.

MVL-CD-MTX is quite stable in storage at 4° C. The pharmacokineticstudies showed prolonged drug exposure by encapsulation inmultivesicular liposomes. The intraperitoneal half-life of freemethotrexate concentration after MVL-CD-MTX administration was 73 fold(39.6 h vs. 0.54 h) longer than that after injection of unencapsulatedmethotrexate.

The total concentration of methotrexate in the peritoneal cavity after aMVL-CD-MTX administration actually increased during the first day andstayed above the original concentration for a period of one week. Thisinitial increase in concentration may be due to differential clearanceof the suspending medium versus the multivesicular liposome particles.

The plasma AUC after the injection of MVL-CD-MTX was similar to thatafter injection of unencapsulated methotrexate (18.4 and 11.2 μM/h,respectively). This indicates that all of methotrexate from MVL-CD-MTXis bioavailable to the systemic circulation.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

I claim:
 1. A liposome comprisingwater, a biologically active, eatersoluble compound encapsulated within the liposome, and a cyclodextrin ina concentration of from about 10 mg/ml to about 400 mg/ml complexed withthe compound within the liposome,wherein the biologically activecompound is released from the liopsome into an aqueous solution at about37° C. at a slower rate than from a cyclodextrin-free liopsome, andwithout substantial compromise to the therapeutic index of thebiologically active compound.
 2. The liposome of claim 1, wherein theliposome is selected from the group of unilamellar, multilamellar andmultivesicular liposomes.
 3. The liposome of claim 1, wherein the watersolubility of the biologically active compound is greater that 1 μg/mlin the absence of the cyclodextrin.
 4. The liposome of claim 1, whereinthe liposome is multivesicular.
 5. The liposome of claim 1, wherein saidcompound is selected from the group consisting of anti-neoplasticagents, anti-infective agents, anti-depressives, antiviral agents,anti-nonciceptive agents, anxiolytics and hormones.
 6. The liposome ofclaim 1, wherein the compound is an anti-neoplastic agent.
 7. Theliposome of claim 1, wherein the compound is an anti-infective agent. 8.The liposome of claim 1, wherein the compound is an anti-viral agent. 9.The liposome of claim 1, wherein the compound is an anxiolytic.
 10. Theliposome of claim 1, wherein the compound is an antidepressive.
 11. Theliposome of claim 1, wherein the compound is a hormone.
 12. The liposomeof claim 1, wherein the compound is an antinociceptive agent.
 13. Theliposome of claim 1, wherein said cyclodextrin is selected form thegroup consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrins,methyl cyclodextrin, ethyl cyclodextrin, hydroxyethyl cyclodextrin,hydroxypropyl cyclodextrin, branched cyclodextrin, cyclodextrinpolymers, and monosuccinyl dimethyl β-cyclodextrin.
 14. The liposome ofclaim 12, wherein said cyclodextrin is 2-hydroxypropyl-β-cyclodextrin.15. The liposome of claim 1, wherein said liposome further comprisesmeans for targeting to a desired location within a living organism. 16.The liposme of claim 15, wherein said means is by coupling with a moietyselected from the group consisting of a sugar, a glycolipid and aprotein.
 17. The liposome of claim 14, wherein said protein is anantibody.
 18. A method of increasing the half-life of a water solublebiologically active compound in an animal in need thereof comprisingadministering to the animal a liposome encapsulating the compound,wherein said liposome further encapsulates water, and a cyclodextrin ina concentration from about 10 mg/ml to about 400 mg/ml complexed withsaid compound; whereby the half-life of the compound is substantiallyincreased.
 19. The method of claim 18, wherein the liposome is selectedfrom the group of unilamellar, multilamellar and multivesicularliposomes.
 20. The method of claim 18, wherein water solubility of thebiologically active compound is greater than 1 μg/ml in the absence ofthe cyclodextrin, and the cyclodextrin forms an inclusion complex withthe water soluble compound.
 21. The method of claim 18, wherein saidcyclodextrin is selected from the group consisting of α-cyclodextrin,β-cyclodextrin, γ-cyclodextrins, methyl cyclodextrin, ethylcyclodextrin, hydroxyethyl cyclodextrin, hydroxypropyl cyclodextrin,branched cyclodextrin, cyclodextrin polymers and monosuccinyl dimethylβ-cyclodextrin.
 22. The method of claim 21, wherein said cyclodextrin is2-hydroxypropyl-β-cyclodextrin.
 23. The method of claim 18, wherein thecompound is s elected from the group consisting of anti-neoplasticagents, anti-infective agents, anti- depressives, antiviral agents,anti-nociceptive agents, anxiolytics and hormones.
 24. The method ofclaim 18, wherein the compound is an anti-neoplastic agent.
 25. Themethod of claim 18, wherein the compound is an anti-viral agent.
 26. Themethod of claim 18, wherein the compound is an anti-infective agent. 27.The method of claim 18, wherein the compound is an anxiolytic.
 28. Themethod of claim 18, wherein the compound is an anti-depressive agent.29. The method of claim 18, wherein the compound is a hormone.
 30. Themethod of claim 18, wherein compound is an anti-nociceptive agent. 31.The method of claim 18, wherein the cyclodextrin is selected from thegroup consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrins,methyl cyclodextrin, ethyl cyclodextrin, hydroxyethyl cyclodextrin,hydroxypropyl cyclodextrin, branched cyclodextrin, cyclodextrin polymersand monosuccinyl dimethyl β-cyclodextrin.
 32. The method of claim 31,wherein the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin.
 33. A methodof treating a pathophysiological state in an individual in need thereofcomprising administering a liposome to the individual, said liposomecomprising a therapeutically effective amount of a water soluble,biologically active compound complexed with a cyclodextrin, wherein theconcentration of the cyclodextrin is from about 10 mg/ml to about 400mg/ml, and the biologically active substance and the cyclodextrin areencapsulated within the liposome; whereby the half-life of the compoundin the individual is substantially increased.
 34. The liposome of claim3, wherein the compound forms an inclusion complex with thecyclodextrin.
 35. The method of claim 20, wherein the compound forms aninclusion complex with the cyclodextrin.
 36. The method of claim 22wherein the liposome is a multivesicular liposome, the compound ismethotrexate, and the half-life of the compound is increased from about18 to about 206-fold over that of a unencapsulated form of the compound.37. The method of claim 33, wherein said liposome is selected from thegroup of unilamellar, multilamellar and multivesicular liposomes. 38.The method of claim 33, wherein the water solubility of the compound isgreater than 1 μg/ml in the absence of the cyclodextrin, and thecyclodextrin forms an inclusion complex with the water soluble compound.39. The method of claim 33, wherein the compound is selected from thegroup consisting of anti-neoplastic agents, anti-infective agents,anti-depressives, antiviral agents, anti-nociceptive agents, anxiolyticsand hormones.
 40. The method of claim 33, wherein the compound is ananti-neoplastic agent.
 41. The method of claim 33, wherein the compoundis an anti-viral agent.
 42. The method of claim 33, wherein the compoundis an anti-infective agent.
 43. The method of claim 33, wherein thecompound is an anxiolytic agent.
 44. The method of claim 33, wherein thecompound is an anti-depressive agent.
 45. The method of claim 33,wherein the compound is an hormone.
 46. The method of claim 33, whereinthe compound is an anti-nociceptive agent.
 47. The method of claim 33,wherein said cyclodextrin is selected from the group consisting ofα-cyclodextrin, β-cyclodextrin, γ-cyclodextrins, methyl cyclodextrin,ethyl cyclodextrin, hydroxyethyl cyclodextrin, hydroxypropylcyclodextrin, branched cyclodextrin, cyclodextrin polymers andmonosuccinyl dimethyl β-cyclodextrin.
 48. The method of claim 47,wherein said cyclodextrin is 2-hydroxypropyl-β-cyclodextrin.